The human genome project, combinatorial chemistry, and assay miniaturization have created a demand for faster, homogeneous methods to characterize protein/protein interactions. This Phase I application proposes to fluorescently label proteins using cell-free translation, affinity purify the proteins using peptide tags, and measure binding affinities using fluorescence polarization. Misacylated tRNA's containing fluorescent amino acids will be used to label proteins at specific amber (UAG) sites, thereby controlling the location and the frequency of labeling. The labeling chemistry will be optimized for fluorescence polarization results and for efficient incorporation of the unnatural amino acid during translation. Ultimately, these methods could be used in drug discovery for screening, specificity testing, structure/function studies, and developing a better understanding of therapeutic drug target pathways. PROPOSED COMMERCIAL APPLICATIONS: The ability to rapidly produce fluorescent protein probes will greatly enhance PanVera's capability to develop HTS assays for drug discovery. Commercialization of the technology in a kit format for probing protein - protein interactions should also generate significant revenue in the research products market.